This invention relates to an analyzer making use of the principle of liquid chromatography and a method of treating the samples for the analyzer. More particularly, the invention relates to a method and an apparatus suited for labeling a sample before separating its components.
Liquid chromatography is characterized by its capability to selectively analyze a specific component after separating the components of a substance in the state of a solution. It is appliable to the analyses of many important items in the field of clinical examinations. On the other hand, liquid chromatography necessitates complicate means of analysis and takes much time for the analysis in many cases. This has been a bar for applying a liquid chromatography to the routine works in chemical or medical checkups such as clinical examinations where a large number of specimens must be treated within a limited period of time. This demerit has been responsible for the delay of spread of the automatic apparatus for this type of analysis.
Analysis of catecholamines can be named as one of the items of analysis which are backward in adaptation to routine works. It is admitted to be a useful item for diagnostic examinations of cellular tumors, abnormalities in circulatory organs, cerebral nervous system, endocrinal metabolism, etc., stress and other morbid states. Therefore, the analysis of catecholamines is attracting attention as a useful and diagnostically important item for group examination of geriatric diseases.
Liquid chromatographic separation and analysis of catecholeamines is generally carried out by reacting the sample with a fluorescent labeling agent and subjecting the reaction product to determination by a fluorophotometer. As for the labeling techniques, there are known two types of method: pre-labeling method in which the sample is reacted with the labeling agent before separating the components by a separating column and post-labeling method in which the reaction with the labeling agent is performed after separating the components by a separating column. The post-labeling method is incapable of high-sensitivity detection because of wide diffusion of components after elusion through the separating column. So that the pre-labeling method is advantageous for determining a trace component in a vital specimen.
As the prior art for labeling (turning into a derivative) catecholamines by the pre-labeling method and subjecting the labeled catecholamines to chromatographic analysis, there are known the methods disclosed in JP-A-61-88148 and 60-143766.
According to the method of JP-A-61-88148, alumina is added to the sample to have catecholamines adsorbed on the alumina while reacting dansyl chloride therewith in the course of adsorption to turn said catecholamines into the derivatives. Then, the derivatives are desorbed from the alumina and the solution containing the derivative is concentrated by evaporation. The concentrated solution thus prepared is poured into the flow channel of a liquid chromatograph for separating the solution into the components and the fluorescence of catecholamine derivatives is detected.
According to the method of JP-A-60-143776, a vital specimen such as serum or urea is injected into the flow channel and then three types of reaction reagents are introduced successively into said flow channel and during their passage through the reaction coil, the catecholamines are labeled (turned into their derivatives). The labeled catecholamines thus prepared are captured and concentrated by a concentrating column and then transferred into a separating column for separating the components and the fluorescence of the labeled catecholamines is detected.
The method of JP-A-61-88148 is unfit for routine works such as clinical examinations. This is because the method takes time for the operations for preparing the sample to be supplied into the flow channel in a liquid chromatograph and also the automation of the method is difficult. On the other hand, the method of JP-A-0-143766 requires a long reaction coil since after the reagents and sample have been conducted into the carrier liquid flow channel, the sample and the reagents are mixed in the reaction coil during their passage through the coil. Also, this method has the probe that it takes much time until the components are completely turned into the derivatives.
Further, in these prior art methods, no regard is given to cooling of the sample before introduced into a chromatograph.